Mitogenome characterization and phylogeny of Huzhu white yak (Bos grunniens) in China

Abstract White yak is a unique and precious economic livestock animal in the world. In this study, the mitogenome of Huzhu white yak was firstly sequenced using Illumina high-throughput sequencing technique and then the assembly was annotated. We also explored mitogenome characterization and phylogeny of Huzhu white yak. Our results showed that the mitogenome of Huzhu white yak is a circular molecule with 16,323bp length including a non-coding control region (D-loop), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA genes and 13 protein-coding genes. The contents of four nucleotides (A, G, C and T) were 33.71%, 13.21%, 25.80%, and 27.28%, respectively, yielding a lower GC content (39.01%) than AT (60.99%). Phylogenetic analysis suggested that Huzhu white yak possessed the closest relationships with Huanhu, Jiulong, Datong, Jinchuan, Sibu, Ashdan and Pali yak breeds, and closer to wild yak and Bazhou breed.

Yaks (Bos grunniens) live in the Qinghai-Tibet Plateau (QTP) and adjacent alpine and subalpine areas (Linnaeus 1766). It has strong adaptability to harsh environments such as high altitude, strong ultraviolet and intense cold and provides local herdsmen with daily necessities such as meat, milk and fur (Wiener et al. 2003). White yak, a unique and precious economic livestock animal in the world, mainly lives in Tianzhu County of Gansu Province and Menyuan, Huzhu and Ledu Counties of Qinghai Province in China (Compilation Committee of animal and poultry records and maps of Qinghai Province 1983). Presently, China own 22 domestic yak breeds including 20 indigenous breeds (Qinghai-Plateau, Huanhu, Xueduo, Yushu, Niangya, Sibu, Pali, Leiwuqi, Tibetan High Mountian, Chawula,Tianzhu, Gannan, Bazhou, Pamier, Zhongdian, Jiulong, Maiwa, Changtai, Jinchuan and Muli) and two improved breeds (Datong and Ashdan) (National Committee of animal genetic resources. 2021). However, except that Tianzhu white yak breed is white, other yak breeds are black. Huzhu white yak (Bos grunniens) was firstly described by Li et al (Li et al. 2021), which resides in Tu Autonomous County of Huzhu in Qinghai Province, China. In our recent study, significant maternal genetic difference was detected between Huzhu white yak population and Tianzhu white yak breed based on mtDNA D-loop sequence variations. For instance, we identified 43 and 3 specific haplotypes in Tianzhu white yak and Huzhu white yak, respectively, and significant genetic differentiation was also found between them (F ST ¼0.2721, p < 0.05) (Li et al. 2021). Therefore, in this study, the whole mitochondrial genome (mitogenome) of Huzhu white yak was firstly assembled and annotated. We also explored the phylogenetic relationship between Huzhu white yak and other yak breeds in China. Our current study would be useful to the genetic resource conservation and molecular breeding programmes of white yak in the future.
Here, an ear sample of Huzhu white yak (Bos grunniens) was collected from Tu Autonomous County of Huzhu, Qinghai, China (101 58 0 N, 36 50 0 E). The voucher specimen (Sample No.: HZ13-20201023; zhijiema@126.com) is stored in the Key Laboratory of Plateau Livestock Genetic Resources Protection and Innovative Utilization of Qinghai Province, Academy of Animal Science and Veterinary Medicine, Qinghai University (Xining, Qinghai, China). The Illumina NovaSeq 6000 platform was used to sequence the whole genome of Huzhu yak with a sequencing depth of 22.63Â.
The mitogenome sequence of Huzhu white yak was submitted to Genbank (Accession No: OK271108). Our results showed that the length of circular mitogenome of Huzhu white yak was 16,323 bp with nucleotides contents as follows: A 33.71%, G 13.21%, C 25.80% and T 27.28%, which yielded a higher AT content (60.99%) than GC content (39.01%). The gene composition, structure and arrangement of mitogenome for Huzhu white yak are similar to that of most   (Clayton 2000;Xu et al. 2015;Hao et al. 2016;Hu and Gao, 2016;Niu et al. 2016;Kamalakkannan et al. 2020;Wang et al. 2021). The mitogenome composed of noncoding control region (D-loop), two rRNA subunit genes (12S rRNA and 16S rRNA), 22 tRNA genes and 13 protein coding genes with lengths of 893 bp, 2527 bp, 1511 bp and 11418 bp, respectively ( Figure 1, Table 1). The length of the two rRNA genes are 957 bp (12S rRNA) and 1570 bp (16S rRNA) respectively, which were separated by tRNA Phe . 22 tRNA genes ranged from 60 bp (tRNA ser ) to 75 bp (tRNA Leu ) and 13 protein coding genes ranged from 201 bp (ATP8) to 1830 bp (ND5). Among the 13 protein coding genes, ATA is the starting codon of ND2, ND3 and ND5, and ATG is the starting codon of others. Three complete stop codons were annotated, i.e. TAA (ND1, COX1, COX2, ATP8, ATP6, ND4L, ND5 and ND6), TAG (ND2 and ND3), AGA (Cytb), and two incomplete stop codons were identified, i.e., TA-(COX3) and T-(ND4) ( Table  1). There are four overlaps in the protein-coding genes, including ATP6 overlaps with ATP8 for 40 bp, COX3 overlaps with ATP6 for 1 bp, ND4 overlaps with ND4L for 7 bp and ND6 overlaps with ND5 for 17 bp. Except for 8 tRNA (Gln, Ala, Asn, Cys, Tyr, Ser, Glu and Pro) and ND6 genes in light strand, other mitochondrial genes of Huzhu white yak were encoded in heavy strand (Table 1). Phylogenetic analysis showed that Huzhu white yak possessed the closest relationships with Huanhu, Jiulong, Datong, Jinchuan, Sibu, Ashdan and Pali yak breeds, and was closer to wild yak and Bazhou breed. However, distant genetic relationships were found between Huzhu white yak and the rest of domestic yak breeds (i.e. Maiwa, Xueduo, Zhongdian, Niangya, Qinghai-Plateau, Yushu, Gannan and Tianzhu) (Figure 2). A further extensive survey of yak whole genome in China is warranted to completely clarify the genetic difference and classification between Huzhu white yak and other yak breeds/populations.

Ethical approval
This study was conducted with the guidelines of the Council of China and animal welfare requirements. Based on the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of China, the Institutional Animal Care and Use Committee of Qinghai Academy of Animal Science and Veterinary Medicine, Qinghai University approved all animal experiments.

Author contributions
Zhijie Ma conceived and designed the project. Sample collection personnel include Jing Luo, Guangzhen Li, Ruizhe Li, Zhijie Ma, Decang He, Wenxian Liu and Yongqing Yang. Jing Luo and Zhijie Ma performed the experiment and data analyses. Jing Luo wrote the original manuscript, Zhijie Ma and Mohammed Yosri revised the manuscript. All authors reviewed and approved the final manuscript, submitted the voucher is Jing Luo and Zhijie Ma.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by the National Natural Science Foundation of China [31960656] and CAS 'Light of West China' Program (3-1).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession no. OK271108. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA791839, SRR17319815, and SAMN24365600 respectively.